Download Bacterial Growth Kinetics of "M. lufu" in the Presence and Absence

INTERNAL IONAL JOURNAL OF LEPROSY ^
Volume 50, Number I
Printed in the U.S.A.
Bacterial Growth Kinetics of "M. lufu" in the
Presence and Absence of Various Drugs Alone
and in Combination. A Model for the
Development of Combined Chemotherapy
Against M. leprae?'
Joachim Karl Seydel and Ellen Gertrud Wempe'
Tremendous progress has been achieved
in the chemotherapy of infectious diseases
during the last 40 years. Today, in general,
every infection caused by bacteria can be
treated successfully with chemotherapy.
One of the important preconditions for this
development was the successful cultivation
of the various microorganisms, thus providing a simple in vitro test system for the development of new drugs. The lack of such
a system is the major drawback in a rational
development of the chemotherapy of leprosy. The only established test system
available is the mouse foot pad technique
22 2". 21
However, this is a complicated
and time consuming test system and includes factors which are difficult to control,
particularly influences of the host organism
on drug uptake, metabolism, and excretion
and therefore on the effective drug concentration for therapy. Besides this, these
pharmacokinetic influences in mice are, in
general, different from the influence of the
human organism on the drug molecules. This
limits the conclusions which can be drawn
for the treatment of leprosy in man. The
described circumstances have led to the situation that only a small number of drugs are
available for the therapy of leprosy. These
drugs are all derived from drugs which have
been used in the treatment of tuberculosis.
Surprisingly, diaminodiphenylsulfone (dapsone, DDS), which is not a strong inhibitor
('.
'
).
' Received for publication on I I May 1981; accepted
for publication on 21 September 1981.
J. K. Seydel, Drser.nat., Dipl.-Chem., Professor
and Head; E. Wempe, Chemical Assistant, Biochemistry Department, Borstel Research Institute, D-2061,
Borstel, Federal Republic of Germany.
20
of M. tuberculosis, shows extremely high
inhibitory potency against Al. leprue in
mouse foot pad experiments 3 ).
In contrast to the practice in the therapy
of tuberculosis, monotherapy, not combined treatment, has generally been applied
in the therapy of leprosy during the last decades. This has created the problem of less
effective therapy and the risk of development of resistant mutants, especially
against DDS 1
2 2
In the early I 970s,
Freerksen and Rosenfeld 1 ) tried to overcome these difficulties. They used a series
of different mycobacterial strains as a model to test the effectivity of various combinations of tuberculostatic drugs. From the
screening I soprodian't, a combination of
DDS, isonicotinic acid hydrazide (IN H)
and 2-propylisonicotinic acid thioamide
(PTH), together with rifampin (RAMP), was
derived and was found to be most effective
against a large number of atypical mycobacterial strains. This drug combination
was then applied very successfully in an
eradication program for leprosy on the Island of Malta. There, for the first time, therapy was interrupted after 1-2 years of treatment. During the first 5 years of follow-up,
no relapses were reported ( 5 •.
(
(
". ". '
'
").
(
MATERIALS AND METHODS
Materials. "M. WU
13 maintained on
Gottsacker medium was used as the test
organism. The culture broth was a modified
Dubos-Davis medium with 0.25% w/v bovine serum albumin fraction V. The authors
are indebted to Dr. F. Portaels, Head, Laboratory of Mycobacteriology, Institute for
Tropical Medicine, Antwerpen, Belgium,
for making this strain available to us.
. '
(
)
50, 1^.S'eydel^Wempe: "M. lufu," a model for M. lepEae?^2 I
Growth. A broth culture was inoculated
from 4-6 weeks old Gottsacker slants into
fresh modified Dubos-Davis medium. To
obtain a uniform suspension, the bacteria
were homogenized in 5 ml of medium
(Potter` homogenittor): the suspension was
then diluted with 15 ml of medium and centrifuged for 4 min at 150 x Part of the
supernatant was taken for electronic counting, and the remainder used to set up the
cultures. Part of the supernatant was taken
for electronic counting and the remainder
used to set up the cultures. The suspension
was diluted to contain —10 5 cells/ml and 50
ml portions were transferred to 300 ml Erlenmeyer culture flasks containing a 2 cm magnetic stirring bar. The inhibitor was added
and the cultures were kept at 31°C. Before
taking samples for counting, the cultures
were vigorously stirred magnetically for
about I min.
Total count (Coulter Counter). Samples
of the experimental cultures were diluted
with particle free saline (0.85%)-formaldehyde (0.2%) solutions, so that a count of
500-20,000 organisms was obtained. Diluted samples were counted with a Coulter
Counter model LB equipped with a 30 ,ctm
orifice. Counts per 50 p.1 were obtained. Instrument settings were: 1/aperture current
1; 1/amplification 1/2; matching switch 40
K; gain 10; lower threshold 7, and upper
threshold maximum.
Viable count (Coulter Counter). Samples
(0.5 ml) from the inhibited culture were taken as a function of time, diluted into 50 ml
of fresh broth (1/101), and incubated at
31°C. Samples were taken after 8 days and
counted with the Coulter Counter as described above.
Sensitivity test. Twelve days after first
exposure to the drugs, the cultures were
diluted to 10 5 counts/ml with fresh broth,
drugs were added again, and the growth
rate determined. To make sure that the cultures were not contaminated, the identity and
purity of the bacilli were checked according
to the usual procedures.
Chemicals (drugs). Clofazimine (CLF)
was obtained from Ciba Geigy, Switzerland; rifampin from Lepetit, Italy; and isonicotinic acid hydrazide, 4,4'-diaminodiphenylsulfone and 2-propyl-isonicotinic
acid thioamide from Bayer AG, Germany.
Trimethoprim (TMP) and pyrimethamine
(PMA) were obtained from Deutsche Wellcome GmbH, Germany. The authors are
indebted to these companies. 4-Cyano-N'phenylsulfanilamide (CNSA) and 4-aminoN '-phenylsulfanilamide (ASA) were synthesized according to standard methods
which have been described elsewhere ('a).
RESULTS AND DISCUSSION
Determination of single drug activity •and
development of resistance, using the bacterial growth kinetic technique. In this paper a
bacterial growth kinetic method is applied
by which synergistic, additive, or antagonistic effects of various drugs and drug combinations including Isoprodian" can be quantified using a mycobacterial strain as a model.
For these studies "Mycobacterium lujit
("), which is not yet completely classified,
was selected. The reason for this selection
was its extremely high sensitivity to DDS,
its sensitivity to RAMP and PTH, and its
low sensitivity to INH, which is ineffective
in the mouse foot pad model (quantitative
data are not available). Thus the sensitivity
pattern of M. leprae resembles more that of
"M. WU than M. tuberculosis (Table 1).
Other similarities are the slow generation
rate (-25 hr) and the maximal growth temperature at —31°C. An additional practical
advantage of "AL In is that it has less
pronounced clumping properties than, for
example, M. tuberculosis. This is a neces-
—
TABLE I. Milli/1111M I /I^C011 Celara
Mil1i11111111 effective dose
(MED from mouse foot pad) f o r various
mycobacteria.
-
io s (MIC) or
MIC^MED'
^
M. tubercul.^leprae
cgIml^µg/ml
DDS"
PTH"
RAMP'
32.0
0.07
0.5
0.25
From Ref. 2.
" dapsone.
isoniazid.
prothionamide.
rifampin.
0.003
—
0.05
0.3
^
MIC
"M.
0.05
5-10
0.60
0.25
^
International .lournal of Leprosy
counts/m1
counts/m1 "M.^PTH
^
M
^
lulu
1982
INH
. control
•
2 pmo1/1
o 4
10 7
A6
A8
■
10
o
12
10 6
V 14 pmol/1
V 18^0^50^100^150^200
t [hr]
FIG. I. 'Typical generation rate curves of "A/.
Wu at 3I°C in the presence of various concentrations
—
of prothionamide (Pill) (electronic counts, total
counts). The curves and generation rate constants k„„„
(in sec ' x 10 5 ) were as follows: ( x) control 7.49;
(0) 7.47: (A) 6.39: (A) 5.73: (0) 4.99: (V) 2.78: (•)
2.26.
nary pre-condition using the Coulter Counter
technique.
In previous papers we have reported on
bacterial growth kinetic studies to quantify
the synergistic action of dihydrofolate inhibitors like trimethoprim in combination
with sulfonamides and sulfones using E.
colt as a test organism ( 1 ". 17 .'""). Other
than the application of special treatment to
overcome the "clumping . ' of the bacteria,
which would prevent reproducible counting
and changes in the nutrient medium, the
same technique as described for E. coil was
used. The number of bacteria in a certain
culture volume is counted electronically
(Coulter Counter) during the logarithmic
growth phase as a function of time. This is
described by the following exponential
equation:
N =^(1)
where N is the number of bacteria per ml
at time t, e is the base of the system of
natural logarithms, N„ is the number of bacteria per ml at the beginning of the experiment, k the generation rate constant, and
t is time. A semilogarithmic plot of the
number of bacteria against time results in
straight lines.
(2)
log N = log N„ + ^ t
2.303
10
0 50 100 150 t
[hr]
FIG. 2. Typical generation rate curves of "Al. /4/ii .
at 31°C in the presence of various concentrations of
isonicotinic acid hydrazide (INT) (electronic counts,
total counts). The curves and generation rate constants k ; ,,,„ (in sec - ' x 10 -") were as follows: (x) control 9.29; (•) 9.29; (0) 9.1(1: (•) 8.9; (A) 8.52: (■)
7.46; (0) 7.11: (•) 6.09: (V) 4.42.
the slope of which is proportional to the
generation rate constant k. Under the assumption that every bacterial cell is multiplying at the same rate, any decrease in
generation rate in the presence of inhibitors
is directly related to the potency of the inhibitor. That means the decrease in genercounts ml^M lulu^ClotaDrntne
105
^
^
0^50^100^150^200^250^300 I [ht]
FIG. 3. Typical generation rate curves of "M.
lulu" at 31°C in the presence of various concentrations
of clofazimine (Lamprene", CLF) (electronic counts,
total counts). The curves and generation rate constants k ; „,„ (in sec -- ' x 10') were as follows for the
first inhibited phase: ( x) control 7.02; (•) 3.81; (0)
3.19; (•) 2.72; (A) 2.19; (■) 1.65; (0) 1.13; and for the
second inhibited phase: (•) 0.0; (0) 0.0; (•) 0.0; (V)
0.0.
^
50, I^.S'eyde/ d Wempe: "M. lufu,'' a model for M. leprae?^23
ounts/m1^"M. lulu"^
10 8
•
o
•
•
tarnplc
control
0005 prnoln
001^•
002^"
005^"
10 7
,^,,,,•,
^,
50 •^00^150 ^•200 ^• 250 l[nr)^300
1,3 5
0
FIG. 4. Typical generation rate curves of "M.
Infir
-
001^
003
at 31°C in the presence of various concentrations
005^007^009
C
of rifampin (RAMP) (electronic counts, total counts).
[prn01/11
The curves and generation rate constants k„,,„ (in
sec -1 x 10 ") were as follows for the first inhibited
FIG. 5. Example of quantitative relations between
apparent "M. hijii" growth rate constant k„,,,, (total
phase: (x) control 8.04: (0) 3.31: (•) 2.20; 0,1 1.84:
(•) 0.081.
counts) and cloLizimine (CLF) concentrations. The
curve is plotted in accordance with equation 3.
ation rate as a function of inhibitor concentration (single and in combination) can be
determined and the fractional inhibition
compared to the control can be calculated.
The inhibitory power of a certain drug can
then be determined by the relation between
its concentration and the related decrease
in generation rate.
Before evaluating the effect of drug combinations to discriminate between their possible additive, synergistic or antagonistic
action, the inhibitory power of the single
drugs was determined. This was done for
DDS, PTH, INH, RAMP. CLF, TMP,
PMA, ASA, and CNSA.
Typical examples for the dependence of
decrease in generation rate on inhibitor
concentration are shown in Figs. 1-4 for
PTH, INH, CLF, and RAMP, respectively.
A range of drug concentration is used
where the growth curves maintain a positive slope. From such plots the activity
constant K,, is calculated by linearizing the
dependence of the decrease in generation
rate on drug concentrations. Two types of
functions are observed: I) a direct relation
between the observed decrease in generation rate and the inhibitor concentration
described by equation 3,
k„ — k„„„ = CK„ + K 1 ,^(3)
and 2) a reciprocal relation (LineweaverBurk type plot) described by equation 4,
I
— K. + K b^(4)
k„ — k„„„^C a
^
where k„ is the generation rate constant in
the absence of antibacterials, k a „„ in their
presence, and K„ and K b are constants. K„
is a measure of the activity of a particular
drug tested and C is its concentration. Figures 5 and 6 show examples of the two types.
022
02^0.6^1.0
1/C
[
1
m
w ud
18
FIG. 6. Example of a quantitative relation between
apparent "M. lidu" growth rate constants (total
counts) k„„„ and dapsone (DDS) concentrations. The
curve is plotted in accordance with equation 4.
24^
International Journal of Leprosy ^
1982
TABLE 2. Activity constant of .some inhibitors of "M. lulu" derived from bacterial
growth kinetic experiments.
K„
awl sec
Inhibitor
4-ASAI3
4-CNSA 13
DDS"
I N H''
Clohtzi mine
0.21
2.12
0.039
RAMP'
0.00027
K„
1- cool^I. sec '
-
0.38
42.5
0.58
292
Conc. range
studied
(pino1/1)
(fint o 1 / 1)
2-50
10-50
0.5-3.0
8-20
0.01-0.1
3-It)
0.003-0.009
6.35 ± 2.25
42.0 ±^12.4
0.45 ± 0.038
15.65 ±^10.4
0.022 ± 0.004
9.68 ± 4.9
0.0024 ± 0.0002
i50
" dapsone.
" isoniazid.
prothionamide.
'' rifampin.
Type I plots (CLF, INH, PTH, RAMP)
seem to apply to bactericidal drugs, type 2
to merely bacteriostatic acting compounds
(DDS, sulfonamides (SA)). The list of K„
values obtained is given in Table 2. For K„
values derived by equation 3 a larger value
means stronger inhibitory power; for K„
values derived by equation 4, a smaller
number indicates stronger inhibitory efficiency. In the case of RAMP, it was not
possible to differentiate on statistical reasoning between the two equations. For
comparison purposes, 50% inhibitory concentrations (i 50 ) of each drug were calculated and are also given in Table 2. The
reproducibility within days for the bacterial
growth kinetic experiments is demonstrated on the example of "M. Wu ' cultures in
the presence of 0.2 p.mo1/1 DDS (Table 3).
There is, however, another interesting
.
TABLE 3. Control rate constants k„ and
apparent first-order rate constants k„„„ for
the growth of "M. lufu" in the presence of
di constant concentration of dapsone (DDS)
in repeated experiments on different days.
The variance in k„ - k„„„ is 0.022, standard
deviation 0.149; with respect to the mean
(2.078) this is ±7%.
DDS^k„ x 10 - " k """^Inhibition
10'
li_tmo1/11^[sec-1]^
[sec -1 1
0.2
0.2
0.2
0.2
8.17
7.86
7.69
8.13
5.91
5.86
5.76
5.97
2.26
2.26
1.93
2.16
27.7
25.5
25.1
26.6
observation in Figs. 1-4. In contrast to the
results presented in Figs. 1-2 for INH,
PTH, and also DDS, where the new steady
state growth rates obtained after drug addition are maintained throughout the total
observation time, the growth rates return
to the control rate in the presence of low
concentrations of CLF and RAMP despite
a very strong inhibition for the first generations. If these cultures are exposed again
to these drugs at the same concentration,
no decrease in growth rate is observed.
This excludes instability or metabolic inactivation of the drug as the reason, rather
it indicates the selection of bacteria with
lower sensitivity against these drugs, i.e.,
the development of resistant bacteria under
the experimental conditions.
Examples for such experiments, where
the bacteria with decreased sensitivity have
been inoculated into new broth and have
again been exposed to the same concentration of inhibitor or inhibitor combinations
as in the first exposure, are summarized in
Figure 7 (see Materials and Methods, sensitivity test). Whereas in the case of RAMP
alone and in the combination of RAMP and
PTH no inhibition of the recultivated bacteria occurs, the cultures remain sensitive
in all other examples of single drugs or drug
combinations. In the case of INH (5
+ RAMP (0.01 ,uM) the inhibitory effect is only observable for about 150 hr.
After this the generation rate approaches
the control rate, but the bacteria remain
sensitive if exposed again to the same drug
concentration. The reason for this is not
known. The reproducibility of these results
Seydel ce Wempe: "M. lufu," a model for M. leprae?
50,
^
25
t ou r to/ml
10 7
10 6
•
,
RAMP^001, 01/1
DOS 02,,no1.
10 5
10 7
•
•
•^.;
/./
10 6
10 5
.^•^•
DOS 02 ^INH Swno1/1
PTH^2 , RAMP 001,101/1
•
•
,
•
9^•
..
. ' •!/'
k,--
ios
•
-•
INN
70
4,--
5 ,^RAMP 001,01/i
100
^
TPTH
200
. -:
2 . PDS 02^NH 5 ^RAMP 001 yrnoin
^
[nd
100
100
200
300
1 [hr}
FIG. 8. Typical generation rate curves of "M.
^
200
FIG. 7. Generation rates of ".11. lulu" in the pres-
(Oil' at 31°C in the presence of isoniazid (INH), prothionamide (PTH), dapsone (DDS) and rifampin
(RAMP) alone and/or in different combinations at the
concentrations indicated.
ence of different drugs at various concentrations as
indicated. (- - -) control; (0) generation rate during
first exposure; (•) generation rate during second exposure. DDS = dapsone; 1NH = isoniazid; RAMP =
rifampin; PTH = prothionamide.
is satisfactory. The calculation of the frac-
tion of less sensitive bacteria present in the
original culture might be misleading because of the fact that we do not deal with
single counts, despite the homogenization
of the bacteria. By the homogenization procedure, only a homogeneous distribution of
bacterial aggregates is obtained, allowing a
reproducible counting.
8 for IN H and PTH and also the % inhibition expressed as decrease in generation
rate compared to the control in Table 4).
More detailed results will be published elsewhere.
The development of resistance can again
be seen, especially against RAMP, in Figures
4 and 9. The strong inhibitory effect is only
operative for 150 hr: after that the genera• M. lulu'
counts/m1
INH %ono( /I
PTH
2 "
DOS 02 "
RAMP 0.01 "
Determination of combined drug activity.
After the evaluation of the single drug activities, the inhibitory power of various
drug combinations was studied. Figures 8-9
show the results with various combinations
of DDS, PTH, INH, and RAMP. Some of
these combinations, at the chosen concentrations, show synergistic effects, i.e.,
more than additive inhibitory effects
(INH + PTH), others only additive or almost antagonistic behavior (PTH + DDS).
The most pronounced potentiation is observed on combining PTH and INH. INH
alone does not show a significant effect on
the generation rate with the concentration
used. It significantly potentiates, however,
the effect of PTH alone (see slopes in Figure
100
200
300
t [hrl
9. Typical generation rate curves of "M.
lulu" at 31°C in the presence of isoniazid
(INH), pro-
thionamide (PTH), dapsone (DDS) and rifampin
(RAMP) alone and/or in different combinations at the
concentrations indicated.
International Journal of Leprosy ^
TABLE 4. Single and combined action of
dapsone (DDS), isoniazid (111111), prothionurnidc and rilampin (RAMP) on
"M. lufu." Concentrations in kunolll; ba•terial growth kinetics technique, Coulter
Counter.
DDS
INII
PTH
RAMP
WI ')
0
0.2
0
0
0
0.2
0.2
0.2
0
0
0.2
0.2
0
0.2
0.2
0
(1
5
0
0
5
0
0
5
0
0
0
2
0
0
2
(1
1
5
0
5
5
5
2
2
0
0
(1
0
0. 0 1
0
0
0.01
0
0.01
(1
0.01
0.01
0.01
0.01
0.026
(1.1)204
0.026
0.0238
0.016
0.0198
0.0201
0.0121
0.0168
0.0116
0.0121
0.092
0.083
0.0147
((.058
0
' ^inhibition
0
21.5
(1
8.5
38.8
23.8
11.5
53.5
35.4
55.4
53.5
64.6
68.1
43.5
77.7
lion rate returns to the generation rate of
the control. The, different degrees of development of resistance are also demonstrated
in Table 5, where the fractional inhibition
in % of the control) is given after 145 hr
and 265 hr. If' these values are compared to
each other, it becomes obvious that, especially in the case of single drugs, a significant drop in inhibition is observed after 265
hr. The exception is INH, but this is probably due to its delayed onset of inhibition.
In general, only the drug combinations
maintain their inhibitory index or become
even better. The most effective combination with constant inhibitory power is the
combination DDS, INH, PTH, RAMP
which, as already mentioned, has successfully been used as Isoprodian" + RAMP in
the eradication program on the Island of
Malta C). The results presented here on
"M. !MU are additional support for the
selection of this drug combination in leprosy therapy. The concentrations used in
the present study are, however, not identical with the concentrations achieved in
plasma under Isoprodian" + RAMP therapy, which, in general, are higher. The concentrations applied in this in vitro study are
smaller in order to maintain positive slopes
-
1982
TABU: 5. Bacterial growth kinetic• results
(Coulter Counter, "M. knit ) for isoniazid
(INH), PTH (prothionamide (PTH), dapsone (DDS), rijampin (RAMP) alone and in
combinations.
-
e% inhihition
a) after 145 hr of inhibited growth
0
0 control^22.9.10 ^counts/m1
23.4.10' counts/m1
0
1^INII
0.68 pg/m1
10.0- 10' counts/m1 55
0.36 pg/m1
2 VIII
3 DDS
((.05 µg/m!
6.5-10' counts/m1 7(1.7
3.9.10' counts/till 82.6
4 RAMP ((.008 prim'
4.3.105 counts/m1 80.0
5 INH + PFH
counts/ml 84.4
PTH^DDS
6 INH
2.1-10" counts/till 90.8
DDS
7^INII
-+ RAMP
h) after 265 hr of inhibited growth (developm. of resistance)
0 control
I^INII^0.68 µg/ml
2 PTH^0.36 µg/m1
3 DDS^0.05 µg/ml
4 RAMP 0.008 µg/m1
5 INH + PTH
6 INH + PT + DDS
7 INH + PTH + DDS
+ RAMP
462 •10' counts/m1
299 -10" counts/m1
264 •10" counts/ml
158 •10" counts/ml
192 • 10' counts/m1
94 •I0 ^counts/ml
17.8•10" counts/ml
2.8.105 counts/mI
35
43
66
58.5
80
96
99
in the bacterial growth curves, a necessary
condition to discriminate and quantify the
effects of the single drugs and their various
combinations by the Coulter Counter technique.
Other possible drug combinations in leprosy therapy. The combination of sulfonamides (SA) with TMP or TMP analogs is
the classical combination where a strong
synergistic effect has been found (I• 7 ) and
this was quantitatively determined using
bacterial growth kinetic techniques (I").
Sulfonamides, however, are weak inhibitors of mycobacterial growth and are therefore replaced in our studies by sulfones
which inhibit the same enzyme as SA in the
de novo synthesis of dihydrofolic acid. Sulfones are strong inhibitors of this enzyme
2 ') especially in M. leprae and "M. 101"
(s). TMP has already been tested using the
mouse foot pad technique. No inhibitory
effect was reported ( 2 1. Because of the discussed limitations of this test system, we
evaluated the inhibitory activity of TMP
and PMA against the model strain of "M.
(
50, I^.S'eydel^Wempe:
"M. lufu," a model for M. leprae?
^
27
TA n LE 6. Inhibitory action of dapsone
(DDS), trimethoprim (TMP), pyrimethamine (PMA) alone and in combination (inhibitor concentration in prnolll) on "M.
counts/ma • M lulu
• control
• DDS 02 pmo1/1
10 7 o IMP 10
A DDS Q2.1MP lOpmo1/1
lufu."
k x 10 -6 % inIsec - '1
hihition
Experiment
no.
DDS
TMP
PMA
(1
0
0.4
0.4
0
0
0
0
0
2
0
2
6.78
6.78
3.89
1.55
0
0
41.7
77.0
161
0
0
0.1
0.1
0
0
0
0
0
5
0
5
7.52
7.50
7.77
5.28
0
0
0
30.0
M174
0
0.1
(1
0.1
0
(1
0
0
0
0
5
5
7.22
7.20
7.22
4.44
0
0
0
38.5
M 173
control 7.64: (0) 7.64; (•) 5.78; (A) 0.67.
la/a." No inhibitory effect could be detect-
0
0
0.2
0.2
0
10
0
10
(1
0
0
0
7.64
7.64
5.78
0.67
0
0
24.4
91.3
M 169
0
0
0.5
0.5
0
5
0
5
0
0
0
0
9.90
9.89
3.11
0.63
(1
0
68.6
93.6
M255
0
0
0.5
0.5
0
10
0
10
0
0
0
(1
9.37
10.2
1^-,-,
___
0.33
0
(1
76.3
96.5
M252
(1
0
0.2
(1.2
0
10
0
10
0
0
0
0
0
0
25.1
96.2
M2
10 6
10
0
50
100
150
200
250 t [hr]
FIG. 10. Typical generation rate curves of "M.
at 3I°C in the presence of 0.2 jimol/1 dapsone
( DIN and 10 pno1/1 trimethoprim (TMP) alone and
in colnhination (total counts). The curves and rate
constants k„„„ (in sec - ' x 10 ") were as follows: (x)
ed even at high inhibitor concentrations
(-^-20 ,uM). It was therefore surprising that,
in combination with small concentrations of
DDS, very pronounced synergistic effects
were observed. If DDS concentrations of
0.2 or 0.5 ,uM are used, decreases in growth
rates of -25% and -70%, respectively, are
obtained. With the addition of 10 or 5 ,uM
TMP or PMA, an almost total bacteriostatic
effect is achieved, whereas these TMP or
PMA concentrations alone do not cause a
significant deviation in generation rate compared to the drug free control (Fig. 10, Table 6). The effect of DDS/TMP and DDS/
PMA combinations is maintained for several generation times. No development of
resistant mutants (less sensitive bacteria) is
observed. The concentration needed to
achieve this effect can readily be obtained
in plasma. Therefore these combinations
could very well be candidates to be tested
in the therapy of leprosy. Further evaluation is necessary.
Differentiation between bacteriostatic and
bactericidal effects. Under conditions where
the drug not only reduces the rate of
bacterial growth but where simultaneous
killing or kill without inhibition occurs, the
applied Coulter Counter technique does not
allow one to differentiate. In addition to, or
instead of, total counts usiniz, the Coulter
7.69
7.69
5.76
0.029
Counter technique, plate counts have to be
performed. This is especially difficult with
"M. Wit," where we have not succeeded
in performing reproducible plate counts.
In a previous paper we have reported on
a kinetic approach, using a modified Coulter Counter technique, to discriminate between total and viable counts 2 "), thus
avoiding the performance of plate counts.
As already pointed out, the generation rate
of bacterial cells in the logarithmic growth
phase can he described by equation 2. If a
sufficiently high concentration of an antibacterial drug is applied to the bacterial culture, a constant number of counts is oh(
28
^
International Journal of Leprosy
tai ( bac te Host a s i s) by the Coulter
Counter in the sample volume (see Fig. 3
for example). Under these conditions, a differentiation between viable and killed bacteria is not possible, however, if the samples
taken from such an inhibited culture at certain time intervals are diluted with fresh
broth in such a way that the concentration
of inhibitor becomes negligible, the resting
bacteria start to multiply again. Only those
bacteria which have not been killed by the
drug start to grow again. The number of
viable bacteria at time t'„, however, is very
small and not countable. In addition, the relative error caused by the number of killed
bacteria compared to the viable bacteria
^50
present in the experimental volume would
be large. After a certain time interval of
new incubation, however, the number of
viable bacteria has increased significantly
and can be counted by Coulter Counter
technique. If the condition holds that the
culture in the "absence'' of the inhibitor
(dilution 1:101) is growing at the same rate
as the control culture, the generation rate
constant of the control (k„) can be inserted
into equation 2 together with the number of
viable bacteria N, determined after a certain time interval t'. The number of viable
bacteria N„ at time l'„, when the sample
volume was taken from the culture and diluted into fresh broth, can thus be calculated. This has been shown to accurately describe bactericidal drug action in the case
of fast growing bacteria ( 2 "). Difficulties
which arise with "M. tofu" are a possible
lag phase after dilution and incubation with
fresh broth (i.e., the living bacteria do not
multiply immediately) and the observed dependence of the generation time on the inoculum size. We have applied the described
method therefore in a slightly modified way
which still may be suitable to discriminate
between bactericidal and bacteriostatic
drug action even if the quantitative aspect
of the kill rates observed is less accurate.
Since only a constant error may be involved, the relative ranking of the effects
of different drugs and drug concentrations
remains correct nevertheless.
In the case of "M. lufu" we have taken
samples from inhibited cultures as a function of time (diluted 1:101) and after a new
incubation for a fixed time interval (8 days)
the viable bacteria have been counted. In
^
1982
counts/m1^M. lulu -^Clofazimine
• control
total cOunls
0.1pmo1/1
• 0 5 "
1.rs1^culture
10
106
viable counts after dilution (1:101) of the
corresponding 1 culture and new incubation for 8 days (Coulter Counter ; A A)
105
'^...^
0
•^•^•^•^1
^100^150^200 t [hr]
FIG. I I. Typical generation rate curves of "M.
hdil at 31°C in the presence of 0.1 and 0.5 timo1/1
.
clofazimine. The curves and rate constants I;„„„ (in
sec ' x 10 - ") were as follows (total counts): (x) control 7.06; (0) 0.56; (•) 0.44; and after dilution (1:101)
of the corresponding Is' culture with fresh broth at various time intervals and new incubation for 8 days the
curves and rate constants k„,,„ (in sec - ' x 10 ") were
as follows (viable counts): (L) 0.0; (A) —5.06.
the case of merely bacteriostatic acting
drugs a constant number should be observed, whereas a bactericidal effect should
produce decreasing numbers. Results for
two clofazimine (CLF) concentrations are
given in Figure 11 and are compared with the
direct Coulter Counter approach (total
counts—see also Figure 3). It is obvious that
CLF acts bactericidally on "M. Wit" at a
0.5 /..04 concentration and bacteriostatically
for the lower concentration (0.1 itM).
When this paper was in the publishing
process, a paper by S. R. Pattyn, et al. ( 27 )
(1981) appeared, which seems to come to
other conclusions on the value of INH,
PTH, and DDS combinations. This is due
to totally different experimental conditions:
DDS concentrations are used in mouse foot
pad experiments where the maximal inhibitory effect is already achieved by this drug
alone.
SUMMARY
Bacterial growth kinetic studies were
performed in a series of potential inhibitors
of M. leprae using "M. tofu" as a model
strain. Reasons why "M. lufu" is considered to be a better model than M. tuber-
50, 1^,S'eyde/ & Wempe: "M. lufu,'' a model Jr M. leprae?
cu/osis are presented. The inhibitory power
of the single drugs has been quantified, the
activity constants are calculated, and the
synergistic, additive, or antagonistic behavior of the combinations is evaluated. It is
demonstrated that a combination consisting
of dapsone (DDS), prothionamide (PTH),
isoniazid (INH), and rifampin (RAMP) is a
very powerful inhibitor of "M. lulu" and
prevents or delays the development of resistance under the experimental conditions
described. This finding is in agreement with
the therapeutic effect of this combination
(Isoprodian" + rifampin) achieved in a leprosy eradication program on the Island of
Malta. Whereas there is no direct proof that
"M. lufu" is the best suitable model for
drug evaluation against M. leprae, there is,
however, nothing in the presented results
which is against this model, especially as
the actions of DDS and PTH or RAMP is
concerned. A new combination of DDS
with trimethoprim (TMP) or TMP derivatives has also been studied and seems to be
a promising candidate. In addition, a technique is described to differentiate between
bacteriostatic and bactericidal action of the
tested inhibitors against "M. lufu."
^
29
RESUME
On a procede a des etudes de la cinetique, de la
croissance bacterienne chez tine serie d'inhibiteurs
eventuels de M. leprae, en utilisant "M. 101" comme
souche modele. Les raisons pour lesquelle "M. lulu"
a ete considere comme un meilleur modele que M.
tuberculosis, sont exposees. La capacite inhibitrice de
medicaments isoles a ete quantifiee, les constantes
d'activites ont ete calculees, et le comportement synergetique, additif, ou antagoniste des combinaisons
a ete evalue. On a pu demontrer qu'une combinaison
consistant en dapsone (DDS), prothionamide (PTH),
isoniazide (INH), et rifampicine (RAMP), constituait
tin inhibiteur puissant de "M. //du," et prevenait ou
retardait le developpement de resistance dans les conditions experimentales decrites. Cette constatation est
en accord avec ('observation d'un effet therapeutique
de cette combinaison (Isoprodian" + rifampicine)
telle qu'on l'a constatee dans le programme
a
d•eradication de la lepre
Malte. Alors qu'il n'existe
pas de preuves directes que "M. lulu" est le meilleur
modele disponible pour ('evaluation des medicaments
contre M. leprae, rien cependant dans les resultats
presentes ne milite contre ce modele, et particulierement pour cc qui concerne ('action de Ia DDS, de Ia
PTH et de Ia RAMP. On a egalement etudie tine nouvelle combinaison de la DDS avec le trimethoprime
(TMP) ou des derives de celui-ci; ce medicament semble etre promctteur. De plus, on decrit une technique
qui permet de differencier Faction bactericide de
Faction bacteriostatique des inhibiteurs audies contre
RESUMEN
"M. /0/.
Se hicieron estudios cineticos sobre el crecimiento
Acknowledgments. This investigation received fi-
bacteriano usando una serie de inhibidores potenciales
del M. leprae, y al "M. lufu" como cepa de prueba.
nancial support from the Chemotherapy of Leprosy
Se presentan las razones de porque el "M. IOU" se
considera tin mejor model° que el M. tuberculosis. Se
cuantific6 el poder inhibitorio de drogas individuales,
(THELEP) component of the UNDP/World Bank/
WHO Special Programme for Research and Training
in Tropical Diseases and from the German Leprosy
Relief Association, D-8700, Wiirzburg, Germany.
se calcularon las constantes de actividad y se evaluO
el poder sinergistico, aditivo, o antagOnico de as cornbinaciones. Se demostrO que una combinaciOn de dap-
REFERENCES
sona (DDS), protionamida (PTH), isoniacida (INH) y
1. BUSI-Iliy, S. R. M. AND HITCHINGS, G. H. Tri-
rifampina (RAMP), es un muy poderoso inhibidor del
"M. Wu" que ademas retarda el desarrollo de resis-
methoprim, a sulfonamide potentiator. Brit. J.
tencia bajo las condiciones experimentales descritas.
2. COLSTON, M. J., ELLARD, G. A. AND GAMN1ON,
E. Drugs for combined therapy: experimental
Este hallazgo esta de acuerdo con el efecto terapeutico
Pharmacol. Chemotherapy 33 (1968) 72-90.
de esta combination (Isoprodian" + rifampina) alcan-
studies on the antileprosy activity of ethionamide
zado en un programa de erradicacitin de Ia lepra en la
and prothionamide, and a general review. Lepr.
Rev. 49 (1978) 115-126.
Isla de Malta. Aunque no hay prueba directa de que
el "M. lufu" sea el mejor model° para Ia evaluaciOn
de drogas contra el M. leprae, en los resultados pre-
3. ELLARD, G. A., GAMMON, P. T., Rees, R. J. W.
AND WATERS, M. F. R. Studies on the determi-
sentados no hay nada en contra del modelo, especial-
nation of the minimal inhibitory concentration of
mente en lo que concierne a la acciOn de Ia DDS y Ia
4,4'-ditiminodiphenylsulfone (dapsone, DDS)
against Mycobacterium leprae. Lepr. Rev. 42
PTH, o de RAMP. Tambien se estudi6 una nueva
combinaciOn de DDS con trimetoprim (TMP) o con
(1971) 101-117.
derivados del TMP que parece ser una alternativa
4. FREERKSEN, E. AND ROSENFELD, M. Fundamen-
promisoria. Ademas se describe una tecnica para di-
tal data, methods and goals of the present research
ferenciar entre la acciOn bacteriostritica o bactericida
on the treatment of leprosy. Z. Tropenmed. Parasitol. 24 (1973) 17 - 25.
de los inhibidores probados contra el "M. lufu."
30^
hiternatioua/ Journal q f Leprosy^
E. AND RosENEEED, M. Leprosy
eradication project of Malta. First published report after 5 years running. Chemotherapy 23
(1977) 356-386.
FREERKSEN, E., ROSENFELD, M., BONNICI, E.,
D i t ANQui , I . , G . AND K Rui , ( „ R _T iiii:Siliz,
5. FREERKSEN,
6.
Combined chemotherapy in leprosy, background
and findings. Chemotherapy 24 (1978) 187-201.
7. 1111CHINGS, G. H. AND BURCIIALL, J. J. InhibiDoti of folate biosynthesis and function as a basis
for chemotherapy. Adv. in Enzymol. 27 (1965)
417-468.
8. KULKARNI, V. M. AND SIADIA., J. K. (In preparation.)
9. LINN', F. AND PIA ERS, J. H. Some characteristics
of the action of dapsone on multiplication of Mycobacterium leprae in the muusc. Lepr. Rev. 48
(1977) 237-245.
10. PEARSON, J. M. II. AND WHEATE, H. W. Dapsone
resistant leprosy. East Afr. Leprosy Bull. 3 (1975)
3-6.
I I. PEARSON, J. M. H., Ross, W. P. AND REEs, R. J.
W. DDS resistance in Ethiopa. A progress report.
Int. J. Lepr. 44 (1976) 140-142.
12. PETERS,
J. H., SHEPARD, C. C., GoRD0N, G. R.,
A. V. AND F.EIZONDER, 1). S. The incidence of DDS resistance in lepromatous patients
in Costa Rica. Int. J. Lepr. 44 (1976) 143-151.
13. PoRTAIA.s, F. Unclassified mycobacterial strains
susceptible to dapsone isolated from the environment in Central Africa. Int. J. Lepr. 48 (1980) 330331.
14. PoRTAEEs, F. Study of unclassified dapsone sensitive mycobacteria isolated from the environment
in Zaire. Ann. Soc. Beige Med. Trop. 60 (1980)
381.
15. SEYDEL, J. K. Prediction of the in vitro activity
of sulfonamides synthesized from simple amines
by use of electronic data obtained from the simple
amines. J. Med. Chem. 14 (1971) 724-729.
16. SEYDEL, J. K., WENIPE, E., MILLER, G. H. AND
MILLER, L. Kinetics and mechanism of action of
trimethoprim and sulfonamides, alone or in combination, upon E. coll. Chemotherapy 17 (1972)
217-258.
17. StADEE, J. K., WEND E, E., MILLER, G. H. AND
MILLER, L. Quantification of the antibacterial action of trimethoprim alone and in combination
ROJAS,
,
1982
with sulfonamides by bacterial growth kinetics. J.
Infect. Dis. 128 (1973) 463-469.
18. SIADEE, J. K. AND WENIPE, E. Kinetics and
mechanisms of action of 'Isolate synthesis inhibitors', alone or in combination, on Eseberichia
coll. III. Pyrimethamine, trimethoprim and sulfamethoxazole. Chemotherapy 21 (1975) 131-145.
19. STADIA., J. K. AND WEMPE, E. Untersuchungen
zum synergistischen Verhalten and zur Pharmakokinetik von Sulfonamid-Trimethoprim-Kombinationen. IV. Eine vergleichendc Untersuchung
zur Potenzierung der I'rimethoprim-Wirkung
durch verschiedene Sulfonamide and einc kritische Betrachtung ihrer Dosierung. Arzneim.Forsch. 27 (1977) 1521-1532.
20. SIADEE, J. K. AND WEMPE, E. A simple, fast and
inexpensive kinetic method to differentiate between total and viable bacteria using Coulter
Counter technique. Arzneim.-Forsch. 30 (198(1)
298-301.
21. SIADIA., J. K., RICDTER, M. AND WEMPE, E.
Mechanism of action of the folate blocker diaminodiphenylsulfone (dapsone, DDS) studied in E.
co/i cell-free enzyme extracts in comparison to
sulfonamides (SA). Int. J. Lepr. 48 (1980) 18-29.
22. SHEPARD, C. C. The experimental disease that
follows the injection of human leprosy bacilli into
foot pads of mice. J. Exp. Med. 112 (1960) 445456.
23. SHEPARD, C. C. A kinetic method for the study
of activity of drugs against Mycobacterium leprae
in mice. Int. J. Lepr. 35 (1967) 429-435.
24. SHEPARD, C. C. Further experience with the kinetic method for the study of drugs against Mycobacterium leprae in mice. Activities of DDS,
DFD, ethionamide, capreomycin and PAM 1392.
Int. J. Lepr. 37 (1969) 389-397.
25. SHEPARD, C. C. A survey of the drugs with activity against M. leprae in mice. Int. J. Lepr. 39
(1971) 340-348.
26. WATERS, M. F. R., PEARSON, J. M. H. AND REES,
R. J. W. Drug resistant leprosy. A comparison
between proven dapsone and proven thiambutosine resistance. Int. J. Lepr. 44 (1976) 152-153.
27. PATTYN, S. R. Activity of the combination of
isoniazid, prothionamide and dapsone against Mycobacterium leprae and some mycobacteria. Arzneim-Forsch. 31(1981) 2155-2157.